JPS62111933A - Bone gestalt forming agent - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

W'μ]1 The present invention relates to the development and growth of bone tissue, or a bone morphogenetic agent which is a peptide that induces growth. More particularly, the present invention relates to bone morphogenetic peptides containing at least the osteoinductively active portions and immunoreactive regions of osteogenic proteins, any of which are of natural, synthetic, or recombinant pNA origin. From the beginning, all the peptide active substances obtained, their production, isolation and amino acid sequences and uses are described. 1 - When transplanted to softer tissue, the bone infiltration (without minerals)
One thing that the applicant must know is that the quality induces the formation of new bone.
C11ll- revealed. When freshly excised bone is treated with cold dilute hydrochloric acid for 48 hours and transplanted into the brain, subcutaneous muscle tissue, or bone defect site, cells in the post-fetal mesenchymal perivascular connective tissue form cartilage and bone. induce differentiation. (llr
ist, H, R1,5science. 150:893-899.1965>This process is-
It is commonly referred to as matrix-induced bone development. Additionally, the body of the inducer was discovered and extracted. It is a substance named bone morphogenetic protein (BMP). U.S. Patent No. 4.294.753, U.S. Patent No. 4.455,256 and 19834f August 1
The application was submitted on the 6th, and the application serial number is 23.
See No. 606. Also, 1llist, H,R
,, et al., Proc, Soc. (-\1” Sad j ko J Jiao [Yu 173:194-199.1
983 and 1llist, H, R,, et al.
Proc, Natl. -〇- Acad, Sci, (LJSA) 81:37 1
-375. See also the 1984 document. these,
Patents, applications, and references are incorporated by reference into this application. BMPs are thought to convert tissue cells into osteoblasts, the cells that manufacture bone. Hypothetically, this process can be classified as a phenotypic transformation, and BMPs can be considered as vacrine molecules. As distinguished from malignant transformation by carcinogens, phenotypic transformation is caused by self-limiting hostage.
This is a developmental process that was carried out by 1 lil J m. Through a process that repeats normal human fetal development, BMPs induce bone IHI follicles, form cartilage, and over several months develop into solid bones. With the use of BMP transplantation by medical practitioners, the traditional 11
It offers significant advantages over subsequent surgery. The list of 41 areas in which 13 MP is currently and will be used includes almost all parts of the body. 1Ill bones destroyed by disease or accident! It can be used for mounting, it can be used to treat victims of scoliosis, it can be used to treat malformed or failed bones, it can be used to repair both ends of a fracture, etc. There is class e. At present, the isolation of BMF) is relatively time consuming and expensive. In order to induce bone formation in vivo, it is necessary to administer 1 unit of BMP, but hormones, vitamins, and metabolites can be measured by administration of 1 gram to 1 picogram (L-body). One objective of the present invention is to identify and produce functional peptide fragments of BMP that result in an increase in bioactivity of 41% per unit of implanted bone-forming biomaterial. Another objective was to create biologically active peptides whose sequences have been determined and then to produce them manually from the constituent amino acids by direct biochemical synthesis, e.g. by the lerrield method. there were. Another object of the present invention is to express a gene by recombinant DNA technology to determine the sequence and mass produce it, or to express a osteogenic peptide by recombinant DNA technology. The purpose was to provide bone morphogenetic peptides for use in constructing screening probes consisting of nucleic acids to identify genes containing the osteoinductive region of BMP.
The characterization of pezotide fragments with F activity is the key to several commercially viable processes for mass producing efficient bone morphogenetic agents. SUMMARY OF THE INVENTION The present invention relates to osteogenic peptides comprising at least an osteoinductive active portion and an immunoreactive region in the BMP molecule. Partial hydrolysis of M F') and
Partial hydrolysis of the non-ml lagenogenic proteins of bone morphogenesis produced bone morphogenetic peptides with relative molecular weights ranging from about 4K to about 7K (Mp). , RMP , -p and named #J, B
It contained osteoattractive and immunoreactive fragments of the MP molecule. 8 and ■ are two types of BMP-1)
, that is, it contains amino acid compositions of Mr approximately 4.1K from bovine and Mr approximately 4.7K±0.3 from human. The first 36 amino acids of the N-terminus of purified human l-B M P-p with Mr of 4.7K±0°3 are given in Table IV. Bone morphogenetic agents containing osteoinductive and immunoreactive regions of the native BMP molecule are shown in Table IV.
Using the amino acid sequence of -RM P -p, it is produced by a conventional peptide synthesis method, such as the Herrield method. The entire or partial amino acid sequence of I3MP-p or Table 1 can be prepared by a technician skilled in genetic engineering to synthesize a DNA sequence encoding expression for the osteomorphogenetic peptide, or It is used to synthesize DNA sequences that can be used as screening probes to isolate genes encoding osteoinductive and/or immunoreactive regions. A synthetic NA or natural gene is inserted into a recombinant vector, which is introduced into a suitable host and
expresses an osteogenic peptide comprising an amino acid sequence substantially homologous to the amino acid sequence of . The osteogenic peptide produced by these methods is disclosed in Patent No. 4,294,753.
: Patent No. 4,455,256: and pending application No. 523, 603, filed on August 16, 1983.
contains osteoinductive, immunoreactive regions of BMP discovered and isolated by Applicants as described in . This invention also uses bone morphogenetic agents containing osteoinductive and immunoreactive regions of BMPs to induce cartilage and/or bone formation in animals and humans, the induction of which can lead to thought. Kell as you can. The present invention further provides a process for the production and creation of bone morphogenetic peptides resulting from partial hydrolysis of pepsin or trypsin of bone morphogenetic protein (r3MP). Bovine BMP-p generated by partial hydrolysis and human BM
P-p was isolated by one or more of the following methods. namely, continuous fractional precipitation gel filtration, hydroxyapatite (HA) chromatography, and high performance liquid chromatography (HPLC). The functional unit of the smallest bovine l'3 Mr]-p fragment isolated by high performance liquid chromatography (HPIC) is approximately 4.
At this time, the smallest active Mr of human BMP-p is approximately 4.7K:i.
It was 0.3. There are many examples of the role of the J's Poor 1 in the Ljpl Gowang' μLMj book Zi body system, ie, hormone production, enzyme activation, myofibril assembly, white fluid coagulation, etc. Flservier Biomedical Pres.
S, New York, N, Y. K,) Pro side n Fol edited by aenick
Neurathll, (1980) pp.
See 501-524. However, neither inactive precursors of [-3MP or BMP-p nor cell membrane sushibuta are known. Without intending to be limited by theory, it is believed that on the way to the formation of the nucleosome, partial hydrolysis of BMP occurs at 1 nvi-Yo due to the action of intracellular proteases, and BMP becomes I ) Induce differentiation of NA. According to this theory, BMP molecules are made up of domains, hinges, and fringes with the active region surrounding a hydrophobic core that is immune to partial hydrolysis. The structure of BMF) is then reduced in size and reorganized, remaining the same or with a stronger bioviscosity.
Generate a peptide core. Based on this hypothesis, an experiment was conducted to generate osteoinductive peptide fragments using Kako Tako 9 through partial hydrolysis. Bovine and human BMP were partially purified and hydrolyzed with pepsin or ]-lipsin, as detailed in Examples 1, II, and 2 below. M
(r approximately 4K to 7K17) osteoinductive and immunoreactive pebubuds were produced. These products are called bone morphogenetic peptides and are designated f3MP-p. Original lI+ other than bovine or human may be used in exactly the same manner. The kinetics of hydrolysis by pepsin includes a rate-limiting step in which the hydrolysis consists of two seven stages. 7th block - Zoo 1, turn, B
Cleaves the fringes and hinges of the MP molecule, leaving behind the active region. In Phase-II, hydrolysis is accelerated and 13 M
P-p decomposes rapidly. For individual badges, B
The densest and largest population of MP-1) molecules reflects the cleavage of 41 different bonds in the hinge and fringe of a single MP molecule, ranging from Mr 4K to 7K, and statistically Mr, which has the highest frequency, is 5.3 ±1.
Leave the active area with OK. Example 1 shows a new bone morphogenetic peptide from bovine BMP (
Example 1 details the production and isolation of BMP-p from human BMP and 1111III1. Example ■ is 0.5M CaCj! An inorganic solvent mixture of 2-6M urea was used to initially extract BMP from bovine bone. Urea has a significant level of 13MP
For bone batches large enough to prepare BMP and F3M P-F, we chose urea first to avoid the relatively high cost of using guanidine hydrochloride (GuHCl), whenever urea is used. ) was found to be blocked, presumably by a strong isocyanate 1-bond, and automated Edman degradation revealed that the N-terminal amino acid of B
Attempts to determine the N-terminal amino acid sequences of MP and BMP-p were unsuccessful. Protecting groups at the N-terminus in proteins cannot normally be removed without decomposing the protein.
It is desirable to obtain BMP and F'3MP-p whose sequences can be determined. This is 'HLG U HCj!
This was made possible by the discovery that F3MP contains an unblocked N-terminal amino acid. Example ■ uses ^purity GuHCj instead of the urea used in Example ■.
! details the use of BMP and BMP-p in which the N-terminal amino acid is unblocked. BMP-p produced by partial hydrolysis of human or bovine BMP with pepsin or trypsin was isolated by a combination of continuous, fractional precipitation, gel filtration, hydroxyapatite chromatography, and high performance liquid chromatography. Various Biological Functions BMP-1) Species Mr'. It was characterized by immunoreactivity with BMP antibodies, isoelectric focusing, amino acid composition analysis, and/or amino acid sequencing. The first 36 amino acids of the N-terminus were sequenced. Example 1 Bovine bone shape 8 protein (bBMP) is described in detail in the previously cited patents, publications, and pending patent applications.
The purification process included the following steps: 1) The bone tissue was decalcified, and the demineralized bone tissue was placed in water in an aqueous solution. A soluble neutral salt is removed and treated with a solubilizing agent for BMP, at which time the bone ragen is changed to 12 Rabun, and BMP is extracted as a solution of the solubilizing agent.Then from the C1 solution. Separate the solubilizer and neutral salt, l'3
Precipitate MP in aqueous solution. In this experimental example r t, urea was used. Purified and partially purified BMP is described below as partially purified 11 M
Partial hydrolysis yields the same bone morphogenetic vegetated product as described in detail for t)-C. Referring to the diagram, FIG. 1 depicts the stages of pepsin partial hydrolysis of bovine BMP. 1g of partially purified B
MP (see Table 1 of Urist et al., 1984), protein of ■
1 μg of pepsin (Sigma Co, SL,
Digested for 2 hours at 37°C in a 21°C, cxN solution containing 1 oz, MO>. Partially purified [3M +1 starting material MIJ, Mr is 2.
It contained approximately 15 weights of proteins ranging from 5K to 90K. After two incubations in pepsin, proteins with Mr ranging from 30 K to 90 K are susceptible to hydrolysis) and subjected to pepsin partial hydrolysis treatment.
The peptide product is 41k for 1k of 3 glues. Group 2 - Hydrochloric acid insoluble/water insoluble Group 11 - Hydrochloric acid soluble/water insoluble group ■ - Chylic acid soluble/water soluble See Figure 1. Partially purified bovine [3M P per ■ protein, 2.
Incubation with 5 and 10 μg (enzyme to protein ratio 1:100) of pepsin for 2 hours in 0.0 IN hydrochloric acid i~
In this case, the separation of hydrochloric acid-soluble/water-insoluble components induced bone formation. Other ratios (1:200 and 1:
500) did not cause this separation. First, after pepsin cleavage, the peptide products were separated into hydrochloric acid iI7 soluble portions (groups ■ and ■) and hydrochloric acid insoluble portions (group L). Group ■ (hydrochloric acid insoluble) is 5 from the hydrochloric acid soluble part.
Hydrochloric acid-insoluble (■ and ■) peptides were separated by ultracentrifugation at 0,0O (l), washed three times with deionized water, and lyophilized. The precipitate (Group ■) was separated from the -t[Mi] (Group ■) by centrifugation at 50.000U for 1 hour and diluted with cold water for 3 times.
Washed twice. Groups ■ and ■ were freeze-dried separately. As noted below, these three groups of peptides generated by partial hydrolysis were further fractionated and 1,4 r
The bioassays were performed using a group of 1, 2, 4, and 7 characters. Molecule kt (Mp measurement is SDS-PAGE
, performed by acrylamide gel electrophoresis. The frozen fraction was solubilized by incubation with 2/I in 0.06 M Tris-HCl (pH 6,8) containing 2 M urea and 0.2% SO8. 5μ! (2,5% IR9/d) of each sample was applied to a 72.6% gel overlaid with a 3% stacking gel and subjected to electrophoresis at 25 mA. The gel was electrophoresed at 25 mA. Water (5:1:
5) Medium 0.25% CooIlassle brilli
Ant b eyes were stained with 1eR-250. The molecular weight is a standard protein product (Pharmacia) with a Mr of 94.000-14.000 and a protein with a Mr of 17.200-
It was determined using a standard peptide of 1.600 (Pharmacia). Relative molecular weight (Mp is the relative migration side light of the six standard substances with respect to the migration distance of the unknown substance,
The logarithm of the molecular weight was calculated by using a blower.
(Swank and f4unkres, 1 9 7
1, Annal, Biochet 3
9 2 4 Bone morphogenetic activity is sw;ss-webs
Determined by implanting lyophilized partially purified peptides into the posterior quadrat muscles of TER mice. Uf (Mr3 of pepsin hydrolysis on the opposite side of the muscular sac-like All)
B, pOp, 24,000, 15,000, 12.00
(1°4.000.8.500.C3,000 and 2,
500 colonies were transplanted. Suspension of new bone is performed by relevant observation of X-ray images, resection, /
j Weight of ossicles still containing water, III orifice Pki, histological body 1 ('! Measurement data, measured by uptake of J,ru4!j6a into calcified new bones) . Histological body J117 measurement 11, using the random point analysis method for the following formula, X of the histologically valid bone method name
Concerning the line image (it's time to go. ・-% II MP-induced bone formation bone shape focusing!
■ It was a good idea. 13 MP
CMRL-(GIB CO) medium. The induction activity per partially purified polypeptide of 0, 1 to 1.0 is low power field.
To count the number of cartilage cells per (low magnification field), J was measured as 3, purified f-3M F) IJ, +
IPlr; 010 minutes tissue culture, S1) etc. 11 lists (CIin, 0rthop)
, 184: 180-187 (1984) Ir.
Repeat 10rItg/d'r three times. In certain rows, almost all of the 13M l) activity was recovered, group T (7E', %) and group ■ (22
%) is explained as the total t1 of the active M. Glue/I [
The [peptide has no I'3MP activity and therefore ψ#
One or more of the characterizations t) could not be performed. As shown in Figure 2, the first two hours of in-4-
The product of protein hydrolysis formed through Zl-based protein maintained high BMP activity. Between 2 hours and 3 hours, HCI! The sum of the soluble part and the hydrochloric acid insoluble product is the partially purified F3 MP starting material.
It's back to the same level of activity. Approximately 1 hour or more of ink, 11 MF) active.
It fell by about 50% to JS. Partially purified BMP starting material has Mr = 24
-V! I) Approximately 15 types of proteins with a range of 2.5K to 90 are present. After incubation in pepsin for 2 hours, proteins ranging in Mr from 30K to 90 were hydrolyzed. 1 for the remaining 7 liters of polype,
M r noil ranges from 2.5K to 30K (1),
Either the partially digested peptides or the peptides reacted with the irrigated peptides.
Although the hydrochloric acid-insoluble segment was four times more sensitive to transplantation than the hydrochloric acid-soluble segment, both sr')s polyj' tallylamide gel patterns showed that proteins with Mr greater than 30 lost w1 in both cases. Therefore, each component is approximately the same in number. Groups T and H peptides were further fractionated as below. Osteoinductive activity in both groups was always associated with peptides smaller than 7K and larger than 3. The lyophilized Group H product was prepared at pl+6.8.
It was redissolved in a mixture of 0.05M FT buffer and 6M urea. This buffer solution was added to Zephacryl S-2
00 column (5cur x 95cm) 1. 'T
Applying a flow control to the tensile flow equation τ', which was controlled by a transport tube, was eluted and collected as five sections. The two sections with Mr greater than 30.000 were combined, dialyzed against water and lyophilized for bioassay of induction of neoformation per grafted protein paste. M less than 30.000
The three sections for proteins with r were combined and treated similarly. Proteins with Mr of 30.000 or less (yo, and further as follows: Hyde[''-1siabatai1~(1-IA)
The fraction was fractionated using 1J mudgraphy. A sample of 80cm was
A column (2,5X40 cm) in 1-716M urea (7) from 0.01 to 0.05, from 0.05 to 0.2,
Elution was carried out according to a stepwise gradient of 0.2 to 0.5M buffer. The proteins were collected into nine compartments, tested by SDS gel electrophoresis, desalted by dialysis against water in membrane sacs with hole size corresponding to a molecular weight of 2.000, and lyophilized 1 Then, transplantation was performed to measure BMP activity. In step 10, the peptides with M's below F were subdivided using Sephadex 0-50 (ultrafine) molecular sieves [1 Mdgr. Phosphoric acid)-1 Dissolved in helium (pH 7.0) and softened with the same helium (1.5 x 100 cm) with a powder ()k. Approximately 20
d fractions were collected, the peaks were combined, cooled, dialyzed extensively against deionized water, and lyophilized. The resulting fractions were analyzed by Swank & Hunkr
es(197p method J:S I]S −PΔ0[
Analyzed on peptide gel. The lyophilized products of Group 1 were dissolved in 100 m 6M urea and subjected to continuous 1-IA reaction as described above.
Purified by Hegelafy and G-50 gel filtration. The subsequent purification of the peptides isolated by chromatography was carried out using the method described above (llrist et al.
al. 1984) and the method detailed in Example 2 below,
Performed by HPIC. The hydrochloric acid insoluble group and hydrochloric acid soluble group of the peptide cleaved by pepsin are
Mr5.6K''-M r 4 containing many molecules of 1,5
K-7K b RMP -1), the smallest of which had a M r of about 4.1K. The pepsin-degraded group q-products, which were fractionated first by Lephacryl S-200 gel filtration and then by HA chromatography, contain a broad spectrum of proteins and peptides. Approximately 95% of the products with Mr below 3 had no BMP activity t'1. Almost all osteogenic activity was present in proteins and peptides with Mr ranging from 2.5 to 30. Plate gel preparative electrophoresis preparations were prepared with HA [171 Hegelafy fraction] and excised from the peptide region of Mr ranging from 4K to 7K and Mr of ie l111 from 2 to 3. A was created. The peptides in the gel sections were eluted using a 6MI probe, and electrophoresis was performed again. A combination of standard protein and peptide products! The Mr assignment of the peptides in the range -C to about 4 to 7K+7) was confirmed. Only components in this range can induce cartilage formation in vitro and in vitro.
Causes bone formation of vo! , :. Peptides with Mr in the range of 2 to 3K or less (+-11M by IA [JM]) did not cause bone formation. Group ■1 of pepsin degradation]-1A [
When performing 171 Hegelahui, Group 1, which was discussed earlier,
(t)-IA chromatography gave the same components (with some different proportions) as obtained. The peptide fraction with Mr ranging from 4 to 7 K is 0.15
It was eluted with phosphate ion at ~0.25M1li1 degree, which was found to cause bone formation4. Peptides eluted at higher concentrations of phosphate had Mr between 1K and 2.5 and did not cause bone formation. The fraction with high BMP activity was separated into 9 subdivisions by pFadex G-50 gel filtration [1]. further) purified with IPI C to approximately 4.1K M
Only one subdivision consisting of peptides with r caused bone formation. By G-50 or HA chromatography (qrel
, -1tl The physically active fraction was separated into 10 subdivisions using a reversed-phase HPLC column.
et al., 1984), a total of 10 subdivisions were bioassayed in tissue culture of muscle connective tissue. Subdivisions containing peptides with a Mr of 4.1K caused cartilage differentiation. Cartilage M4t showed differences in 2-9% of the total tissue area in the six rapid fractions of culture. The other 9 subdivision orientations with higher and lower Mr components did not cause chondrocyte differentiation in the other 27 cultures prepared under the same conditions. ) The pI of the 4-7K Mr peptide isolated by IA chromatography and IIPI-C is 15oe
electric F'ocusing, Amst
erdam, Nortt+11o11and, P
ubl, CO,, 1976 old ghet mouth and Dr
The pH of one cooling gel (5% polyacrylamide and 2% ampholite) was measured using a microphone npH nail by isoelectric focusing according to the method of ySda18. Focusing was performed at 2°C for 3.5 hours at 10W. DI was approximately 3.9. Amino acid analysis is performed using Amino Acid Analysis i1 (Modify
dBoclvanl 21 ). Cystine and hemicysdine were added after conversion. Table 14.
2. The composition of the bovine 1'3 M P-ρ4.1K peptide. p(7)
/'mi/acid analysis amino acid nHo1es H
ole% Res/41Res putative residue lysine
5.28 5.26 2.16 (2
) Histidine 2,96 2.951.21
(1) Alkynine 566! +
, 64 2.31 (2) Asparagine @13
゜01 12.97! 132 (5) Threonine 3.93 3,92 1.61
(2) Hirin (Corr. bV7.5%) 4.68 4.671.91
(21 Glutamic acid 17.98 17.93
7.35 (7)-/Lorin 657
6.55 2.69 (3) Glycine
5.82! 1.80 2.34
(2) Alanine (5,086,062,
48+2-3) 1/2 Cysteic Acid 5,12 5.
10 2.09 (2) Valine 4.
92 4.91 2.01 (2) MethiAmine (0,94) -(0,38) Isoleucine 3.39 3.38 1.39
(1-2) Leucine 6.88 6.8
6 2.81 (3) Dirosin 4
．． 78 4.77 1.96 (2) ■
group and Mr4 to ±1.5 to Mr5.6 of the knockout peptide
．． The 1K peptide was further purified by hydrophobic column chromatography and analyzed on an air/liquid phase protein sequencing instrument (Hewick, et al., 1981, ).
', Biol, Chei, 256:1990)
, Mr4. of marginal proteolysis by pepsin. IKBMP
l) The product had a blocked N-terminus, which would be identical to that of Mr18.5K BMP. To test the immunological activity of bovine BMP-ρ using dot ELISA, Hawks et al.
Biocell, 119:142-147.198
2), we found that it cross-reacted with the mouse antibiotic BMP (Mr18,500>. In this way, bBMP-p
The immunoreactive and biologically active fractions of (Mr4.IK) and bBMP (18,5K>) were found to be identical. The degraded low to high M of the components of groups ■ and ■
To fractionate r-peptides, ultrafiltration using MrlOK pore size was used. From 120 implants (1-10 mg samples) and 10 1511 g samples of partially purified BMP-1' (33 partially purified BMP-1') (triplicated) and 1511 g samples occupied by induced osteogenesis, The percentage of muscle was calculated as h1. Partially purified 13MP replaced 50-90% of the quadriceps muscle. The limit pepsin proteolysis product soluble in 0.01N HCII/insoluble in water for group ■ is 20-7
5% replaced; 0.01N HCJ of substitution-pu! 50-84% for insoluble/water soluble; 0.01N for group ■
0% for HC7 soluble/water soluble, 1-1 for deposited stones
It was directly proportional to dose in the range of 0 IItg. 10q
The lower doses were found to form more bone in the quadriceps compartment, including and producing deposits that cross over from the pelvis to the opposite leg. Continuous HA chromatography and S-200 or G-5
Partially purified BMP-p (+1.5K to Mr5.6) from group ■ or ■ intermediated by 0 gel filtration
>5Rg transfer Ili' caused bone deposits occupying 32% of the trough of the quadriceps muscle compartment. A detailed histological investigation revealed that the IC bone did not contain muscle tissue, and the amount of wet weight deposited in the IC bone was approximately 200 #Ig.
P-p in tissue culture (causes soft H differentiation,
It was shown that this is the minimum intermediate polypeptide structure that determines biological activity. The mechanism of marginal pepsin proteolysis of 1A1hi1-[-3Ml) (hF(MP) affects the Mr of 4K-7K and the osteoinductive and immunoreactive properties of f3MP). A peptide of MRU was produced. Add 1 solution to IM 011 H of pepsin protein autolysis product at 8-20C.
) By gel filtration, peptides with 4K...~7Kf7) Mr were separated from high Mr and low Mr polypenods. f! Induction activity f21 is rebe U' 4K...7Kri Mr.
I was concentrating on the right PeiJdo and -C. Nothing was found in the fractions with Mr above 7K or below 3.
,two. For G −h (′) filtration of these fractions, J, S,
A group of peptides with a Mr of 5.5 ± 0, 8 K and 11 were single 35-M. This group induced cartilage and bone differentiation in mouse thigh electromyography (142). G50 middle separation/Ch f3 M I]-[) 1-1
1) 11i in history with t C! A J-peptide with an Mr of 0.3 on 4.7K was prepared (q).
Approximately 1, F'1 key 1'l fresh wet bone research l
) - Biochemistry obtained 4.7K, E], OK, and h RM P -p with Mr of 4.4. 4°7K Hi1-11M P-[) formed cartilage in tissue culture]. The test and method are described below: Anatomical G1ft5 Act of C
At autopsy of a 22- to 51-year-old Nori IM within 12 hours of accidental death, bone cortices of 1 to 5 kg were excised according to the A1Ifornia guidelines. The anterior trunk cortex was extensively lavaged, mechanically demyelinated, and 1:1 cloned [1].
1\Remove fat with methanol, freeze in liquid N2e, and crush into particles of 0.5#lII+3 using WileyHill]. - The crushed base particles are 0,
Decalcified for 48 hours in C3N HC1, washed,
4M guanidine containing sulfhydryl group enzyme harmful agent
C1(G(1[4C1>-Ch[3M]) was extracted and fractionated precipitation was performed with 1,5 M GuI-I CI containing 5 mi+oles/1 of N-Tyl 11 neuamide. 0.2% Male JA nectin was removed with X-100, and matrix G (ja protein (MG
rlO. 0 (10 removed. One gram of protein insoluble in 1.5 M Gut-ICI was added to pepsin (Sigma Co,
St. 1 ouis) 10μ"J/IRg<ratio of enzyme to protein 1:100) of 0.01N Ll (1!) was decomposed in a solution for 2 hours at 37°C. 11C decomposed with pepsin
I-insoluble/water-insoluble and)-I Cj! The soluble/water-insoluble fraction was centrifuged at 50,00051/hour to obtain HC1
Separated from t+f soluble/soluble water soluble white. Hydrolysis is 0
．． Dialysis was carried out by increasing the pl+ to 7.0 with 0.01M NaOH and 5 pectrophors.
tubing (hole rise omitted, molecule!yi 2, 0
00 Spectrum Co, los Angels
es. C△) Termination by dialysis against cold water. 0, ()IN H(1! Lyophilize the soluble product,
Measure the weight and find it f7)0. 7f/, 4 MG
'JH (C, pH 6,8+7 in e) dissolved in 0.5 MIJ acidic acid solution (a). This buffer solution is applied to an L horn 7cryl S-2Op color lx (5 atr x 95 ctrr) flowing downward,
It was regulated with a bamboo pump and collected into 5 fractions. 3(1
The two fractions with Mr greater than or equal to 0(')0 were collected, dialyzed against water, and freeze-dried again for bioassay of new bone formation generated against the implanted protein. 30゜0
Three fractions with Mr less than 00 were collected and prepared in the same manner. 80 to 80 protein samples with Mr below 30,000 were subjected to hydroxylated phosphoric h column (1”△) (2,5
x4.0cta>, 0.01 to 0.05.0.0
h~('), eluted with a stepwise gradient of 2,0.2-0.5M 111 degree phosphate buffer. The protein was collected into nine fractions, probed by s r] s gel electrophoresis, and reduced to fluid sacs (pore size, molecular weight 2,000 by dialysis into cold water).
The cells were desalted in 0), lyophilized, and transplanted to examine BMP activity. The peptides (with Mr less than or equal to 10) were subfractionated using a [I Fadex G 50 Ultrafine Traction II Rashibook [J Matgranoy]. Freeze-dried peptide rimples containing 4 M G LJ HC axes of 0.01
Dissolved in M putrium phosphate (11117,0),
Column equilibrated with slow i solution (1,5X1()Ocm)
Nika()ta. Fractions of approximately 20 mm) were collected, and each peak was combined, thoroughly dialyzed against cold IJ1 deionized water, and lyophilized. (7) I, the two fractions,
Swank & Hunkress Method (1971) 1
, ZJ(V)Sl) 5-PAGE analyzed using peptide gel. 0,01N HCJ degraded by pepsin, remaining after 2 hours of marginal pepsin proteolysis! The insoluble, water-insoluble product was centrifuged at 50.000 g to yield ('), 0
1N HOj! Separate from soluble products and cool 1e
t t, t Wash three times with deionized water and collect by lyophilization.

[Nota. Freeze-dried proteins and peptides
Dissolve in M G LJ HCI and fractionate in the same manner as above! , :. Matrix q1a protein maintains insoluble Bamboo synthesis with high F3M+) attachment
It was removed by solubilization in M G LJ l-I CI and superfiltration. Reversed-phase high performance liquid stream [1 MADROGRAPHIE (HI”' CI
-) was carried out on the protein fraction of Mr5,500:jl, 0Op isolated by Sephadex G50 [171~graphie.
These fractions were dissolved in 4M guanidine hydrochloride that was M-blocked with ). The Lean-sol, which was centrifuged using a Noi [1-66 membrane (to remove microscopic insoluble proteins)] was transferred to a Vydac (C4) column (4.6 m x 25 cm).
m) (Co, C1ty) crab ('', 0.1% trifluoride) 4-n acid-resistant aqueous solution ('I''F' A)
and 0.1% l'FA in a[[-2]-lyl solution (
It was eluted with a gradient of AN). Gradient is 20% ANC star 1
~ and continued at 35% AN for up to 15 minutes (1% per minute). 35%AN to 50%ΔN Yogu is 10
The column was then re-equilibrated. 1-FA with a high concentration is useful for better separation of closely eluting substances and is useful for analytical chromatography and solar chromatography [171- This gives a better correlation between Noy and λ. After collecting the appropriate fractions, the fractions were removed by centrifugation. Rimples were collected in a dry state by freeze-drying. For determination of Mr, protein fractions were examined by notrium dodecyl sulfate polyacrylamide plate gel electrophoresis (SpSPAGF). iii solubilize the freeze-dried protein,
0.06M Tris-H with 2M urea and 0.2% SO8
CJ (1186,8) 24 hours in" - 11 bet. 5 microphones each [1 liter (2,5Rg/
The 1J sample of d) was run on a 12.6% gel with a 3% standing gel and electrophoresed on a 251TIA IC, 0.25% Coomaissie Brilliant gel.
- Plue R-250 methanol/l'li acid/water (5
:1:5) 31 molecular weight of gel stained with solution Ha, Mr.
Protein standard material in the range of 94,000-=14.0()0 (Pharmacia) and Mrl 7.20
Peptide standards 1i4 (Ph
arllacia) was used to determine the molecule. 6
By plotting the logarithm of the minute f tail for the migration vtI separation of the embolistic peptide, and comparing the distance of the unknown ripple, the relative molecule ffl (Mp is calculated by η1 /= (S
wank & hunkress). Todan secret map of human BMP-p molecule; L, M r 5
, -C was concentrated in the region of ±0.8 to 5. Example I
BMP-1) likewise contained Mr molecules of 4.1K+:0.3. Bioassay of BMP-p for osteoinductive activity
Determined by implanting 128 lyophilized samples into the hind leg and buttock muscles of w1ss-Webster a-/mouse. ] for l-rl-le, 10 samples of osteonectin (Mr38.000) each.
, Mr24,000. MGf) (Mrl 4.000~
15.000), and Mrl2゜000.8,500,
4,000,3. Pepsin proteolysis of (')00° and 2.50 (')! 1 degree new bone suspension was transplanted into the contralateral hind leg buttock myocardial muscle, as described in Example 1, to avoid correlation with the X-ray h'' true observation, it was deleted! Bone dampness, histological section,
Histomorphometric data were measured by the uptake of 45Ca into iodinated new bone. histomorphological it
1IQ data is analyzed to random point 1
7. Hi1~RMF'-p is CM containing 15% beef tallow serum
Rl-(G I F3CO) is soluble in culture medium and 10
in tissue culture with 10 μg/- of HP L, C fraction,
It was quantified in triplicate by the 5ato and ur+st methods. Table H summarizes the results of bioassays of osteoinductive proteins and peptides containing secondary F products at various stages of purification. Bone matrix non=] Lagen-like protein (Gutl (C
I solution (separated by fractional precipitation) had low levels of osteoinductive activity. (Partial purification by 1Δ chroma 1 by chromatography increased the specific activity by 25× as measured by histomorphometric measurements, and the right calcified new bone had 45 Ca
The image size was increased to 950× by incorporating . Further purification increased bone formation in both parameters. Table ■ Comparison with peptides that have been reduced by limit pejcin proteolysis, the protein's yield is 1! Its intake Cpm/#I7 Transplant protein 1.5M GullCI! Insoluble/water-soluble non] lagen protein. 90~30K O,5302+-H)43
8 to Tri-1 to X-100 soluble protein 0 20
0934K CL-61'3 isolated AsteA nectin, O25±1130 + 24
+ 22 K 1.5M Gut-(Cj! Soluble protein 0 31±
112/IK hydroxylated phosphate non-binding protein +1@0 26shi0817.
BHP+15 on 5 → q1a protein on 14 12.4 2660±
50014KIIQj! a protein 0 °
45±10 polypeptides of 7 and 14 treated with pefcin 28.5 3696±
9805.5±0.8 to G-50 Nakamen polypeptide 13.0 2964±
6084.7KIIPLc Purified Tissue Culture Polypeptide Cartilage N,D
. HA RIV t-'J -y 7 Eat HP I
-CnyoVJ if Ill 01 of the peptides were placed in a micro npH needle (Hicroelec) for lltl measurements on cold gel (5% polyacrylamide and 2% ampholite)
Righetti using trode, Inc.)
and Drysdale (1976), determined by isoelectric A-casing. Focusing was 10 W for 3.5 hours at 2°C. Mr4.7K17)
The pI of the peptide was approximately 7.1±0.1. For amino acid analysis, use 1←modHied8 for amino acid analysis.
acka+an 121). Analysis of cystine and hemicystine was performed after performic acid oxidation. The composition is
This was characteristic of neutral peptides. The amino acid composition of the Mr4.7K peptide is shown in Table ■. Table ■ Mr4.7K isolated by reverse phase 1-1 +) l G
Amino acid analysis of L:l-BMP-p Ser! 1.08 442.3! 1
．． 47 2.78 (3) (corr iy 10%) GILJ 18.79 2426.0 20
．． 23 10.27 (10) Pro 7.
54 732.2 8.12 4.12
(4) Gly 2゜16 123.2
2,33 1.18 (1) Ala 6
．． 24 443.5 6.72 3.41
(3) 1/2 Cys 6.30 3.3
1.35 3.1 (2) (Cyst
eic acid) Val 4.18 414.4 4,5
0 2.28 (2) Met 1.26
16!1.3 1.36 0.69
(1) 11e 4. (104!12.6 4.
31 2.19 (2) 1-eu 4. i
i8 529.5 5.04 2.5B
(2-3) Tyr 5.32 868.1
5,73 2.81 (3) Purified peptides in a gas/liquid phase protein sequencer +'P
Analysis of the amino acid sequence of LC. Table ■ shows l-1P made from human f3MP-p.
Partial amino acid sequence of i-C(7) N-terminal segment 1 is shown. The amino acid sequence of the first 15 residues of the N-terminal segment of J0hsMp-p was sequenced on three separate bone batches. was completed. The sequence of residues 17-27 was determined with less certainty, so 2B-36 was determined with an interesting discrepancy that required special investigation. = 48-Hitoichi Partial amino acid sequence of hBMP N+-12-ILE-PRO-GLN-GLN-ARG
-ARG-TRP-ARG-ALA-1-YSll
12 13 14 15-VAL-GI M-
ASN-ARG-IL-E (1-15, J:Li
16 17 18 19 20 less certainty) -(ALA?)-ARG-ASP-SER-TY
R-LYS-PRO-VAL-Hls-GLU2
6 27 128 29 3O-LELJ-AS
N-IARG-CYS-ALA (from 17-27
31 32 33 34 35 little nai lj [
f Actual K) -ASP -G LY -TYR
-ARG -LE LJCYS- 1 49 - N! U4III Tryptic proteolysis of purified or partially purified BMP yielded BMP-P. B induced pepsin
The trypsin induced F3Ml)-P was purified, bioassayed, and characterized by the same method reported for the Miml of M P -P. Partially purified raw BMPlg was treated with trypsin (T
Bovine pancreas treated with PCK, Sigma. st, Louis) for 15 min, 30 min, 1
time and 4 hour intervals at 37°C. IMTr
5/g total protein at ts pu 7.20
Digested Bg. At the indicated interval, ρ111.0
The hydrolysis was terminated by adding IN-HC1. The solution was centrifuged for 10 minutes at 20,000 rpm to separate the acid-soluble precipitate from the acid-insoluble precipitate. The precipitate was washed three times with cold water. Pour the acid-soluble-F purification solution into Sρectrapor piping (pore size 2,000
MW, 511eCtrllll, CO,,1,os^
geles) and dialyzed against ionized water (three times). The water-incurable precipitate formed inside the sac was centrifuged at 50,000 g for 1 hour at 7'C, then the precipitate was washed three times in deionized cold water. and separated from water-soluble components. The water-soluble supernatant and washed precipitate were separately concentrated, lyophilized, and weighed. The lyophilized product was weighed and redissolved in 0.05 M Phosphate Buffer-8-4 MG U HCl applied through a 200 gel filter, pH 6,8. 5ephacryl S-200: 5ephacryl S-200: regulated by a screw pump and collected in 5 fractions
The solution was applied to (5 crtrx 95 cttr). Two fractions with Mr of 30,000 or more are pooled,
11 proteins that were dialyzed against water and transplanted! ! To J? Lyophilized again for bioassay of induced bone formation. Three fractions with Mr protein below 30°000 were pooled and similarly prepared. Proteins with Mr less than 30.000 are 5eph
Adex G-50 (1lltrafine) was subdivided using molecular fluid VS fractionation. A swatch of the lyophilized polypeptide was added to a 0.0.
The mixture was dissolved in 0.01M sodium phosphate and yarned into a ram (1.5 x 100 cm) equilibrated with the same buffer. Fractions of approximately 20 mft were collected, tied together inside each tip, extensively dialyzed in cold deionized water, and lyophilized.
Pay 1 no for wank and Hunkres, S[)S-
[)8G EE Bubd analysis. Subsequent purification of the IIIC polypeptide by G-bO gel passage was carried out on a hydrophobic column (1lltr
apore, reverse phase Spherogel, Beclva
completed by H l) I C using n). Implantation of partially purified I[f3MP] induces cartilage and bone formation in mice:. This deposit replaced approximately 4% of the volume of the quadriceps compartment. Implantation of trypsin-limited proteolytic products - both acid-insoluble and acid-soluble fractions - induces cartilage and bone tissue that fills the entire oral compartment, even sometimes extending across the midline, resulting in IC0 acid Reaction with insoluble or water-soluble proteins may result in the formation of a single-wire synthetic fiber.
Ginai. J for transplantation of limited tryptic proteolytic products;
The bone deposits induced by this method showed the same structure as the deposits produced by BMP implantation. This bone consisted of a lamellar bone envelope filled with bone marrow and a woven bone core containing cartilage and chondroid bone inclusions. The ITV bone morphogenetic agent is synthesized by conventional chemical synthesis with an amino acid distribution of RM P-P as shown in Table IV.
(or essentially the same equivalents). The chemical synthesis method for peptides is He
This is the rrifield method. Herrifield, R.B., J., Ch.
ew, Soc, 85:2194.1963:
Herrield, R.B. 5science, 150: 178.1965゜This J, a synthetic osteogenic amorphous peptide agent, is a natural [
Contains the osteoinductive and immunoreactive domains of IMP. Example V By methods known to those skilled in the art of developmental engineering, a synthetic pNA rgene for expression for bone morphogenetic agents was encoded as shown in Table rv.
It is composed of the amino acid sequence of -P and the 4-ton sequence. The required NA sequence is determined from the amino acid sequence and the developmental code, and the "gene" is chemically synthesized and inserted into the exchangeable vector. A recombinant vector is introduced into a host cell, such as E. coli (Obiki 1), whereupon the vector is replicated and expressed when the cells are cultured. During growth, the host cell machinery transcribes the mRNA from the synthetic gene and translates it into the bone morphogenetic peptide agent. Technologists perform vector selection, host selection, and location of the synthetic gene within the recombinant vector. Variables such as are used to manipulate the in vivo culture of native 13M P or BMP-P, which is an effective factor in the production of bone morphogenetic peptide agents that exhibit osteoinductive and immunoreactive activities. For one of these methods used by humans skilled in the art of genetic engineering, see the Examples, Haniat et al.
is, T,, Frltsch, E, F, and 5alIbrook. J,, (19B2) Ichikano Noriko 1L8 1abo for residence service
ratory Manual, Cold Sp
ring 1laboratory,
Co1d Spring, Harbor New Y
(see above), the bone morphogenetic agents are the BMs shown in Table IV. F)
A cDNA containing part or all of the two-tone amino acid sequence for P is produced by expressing a cloned CDNA or a cloned segment of vertebrate DNA. Using known recombinant DNA Q techniques, a segment of the vertebrate genome or CDNA was inserted into the vector, and the lfl recombinant vector was then inserted into the host J-'s body. Introduce it inside. Novel radiolabeled nucleic acid block 1 of heavy chain DNA or RNA containing some or all of the amino acid sequences of tBMP-P shown in Table TV.
-1-b is chemically synthesized. These probes (or complementary twists of these special acid-containing molecules) sift through the clusters of preferential sequences <-j L, thereby promoting osteoinduction of B M F). The selected population is osteogenic or anti-BM.
Peptides are cultured and tested for production of peptides that are immunoreactive with P antibodies. A novel radioactive elf-sensitive nucleic acid probe containing 1 ton [for some or all of the amino acid sequence of 3 M P -P] shown in Table IV includes 1 ton for the osteoinductive region of 13 M I) Used for identification and selection of vertebrate DNA RNA (5cl) by NA or segmental hybridization. Those selected are used to construct recombinant vectors for the production of bone morphogenetic agents in a host. Although the urgent technical content contains essential information related to the invention, a number of the publications cited herein may be helpful in understanding the background of the invention and the state of the art. Seem. Accordingly, the cited publications in Libet are hereby incorporated into this document as relevant to the present technical content. It will be understood that this feature listing and technical content of the invention is intended to cover all modifications and improvements of the invention that are within the spirit and scope of the invention. inserting amino acids within certain BMP-p amino acid sequences that do not essentially affect the bone morphogenetic activity of the molecule;
Deletion or substitution is within the skill of the art. The invention is broad enough that it is specifically stated that it includes intentional deletions, additions, or substitutions. Additionally, it would be possible for those skilled in the art to produce such improved proteins recombinantly.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the limited proteolysis of bovine osteogenic protein by pepsin, and FIG. 2 is a graph showing the decrease in osteogenic activity due to pepsin proteolysis.

Claims (1)

[Scope of Claims] (1) Said peptide obtained from whatever its origin, including natural, synthetic, or recombinant DNA, i.e., osteoinductive activity similar to bone morphogenetic protein (BMP); A bone morphogenetic agent containing peptides that exhibit (2) the terminal portion of the BMP has a relative molecular weight (Mr) ranging from about 4 Kb to about 7 Kb, and the peptide agent is whatever the N- of bone morphogenetic protein (BMP)
The bone morphogenetic peptide agent according to claim 1, which includes the amino acid sequence of the terminal portion. (3) The following amino acid sequence: -ILE-PRO-GLN-
GLN-ARG-ARG-TRP-ARG-ALA-L
Patents characterized by containing YS-VAL-GLN-ASN-ARG-ILE and encompassing additions, substitutions, deletions or insertions of one or more amino acids resulting in a peptide similar to the osteoinductive activity of BMP The bone morphogenetic peptide agent according to claim 1. (4) The following amino acid sequence: -ARG-ASP-SER-
TYR-LYS-PRO-VAL-HIS-GLU-L
The bone morphogenetic peptide agent according to claim 3, which includes EV-ASN-. (5) The following amino acid sequence: ARG-CYS-ALA-A
The bone morphogenetic peptide agent according to claim 4, which includes SP-GLY-TYR-ARG-LEU-CYS-. (6) Derived from proteolysis of BMP, approximately 4K to 7
The bone morphogenetic peptide agent according to claim 1, which has a relative molecular weight (Mr) of K within a range. (7) The bone morphogenetic peptide agent according to claim 6, wherein the proteolysis of BMP is performed using pepsin. (8) The bone morphogenetic peptide agent according to claim 6, wherein the proteolysis of BMP is performed using trypsin. (9) The bone morphogenetic peptide agent according to claim 6, 7, or 8, wherein the proteolytic BMP is human BMP. (10) The human BMP-p is approximately 4.7K±0.3K
The bone morphogenetic peptide according to claim 9, having a relative molecular weight (Mr) of . (11) The bone morphogenetic peptide agent according to claim 6, 7, or 8, wherein the proteolytic BMP is bovine BMP. (12) The bone morphogenetic peptide agent according to claim 11, wherein the bovine BMP-p has a relative molecular weight (Mr) within the range of approximately 4.1K. (13) Claim 1, wherein the peptide is derived by direct chemical synthesis from the constituent amino acids, and the peptide includes the amino acid sequence of BMP-p, including the addition, substitution, deletion, or insertion of one or more amino acids. The bone morphogenetic peptide agent described. (14) The expression code of the peptide is BMP or BMP
Codon sequences similar to the osteoinductive activity of -p, natural, synthetic,
Or the DNA obtained from any origin including semi-synthesis
The bone morphogenetic peptide agent according to claim 1, which is derived from the expression of a DNA sequence comprising the sequence. (15) The expression code for the peptide is the following amino acid sequence:-
ILE-PRO-GLN-GLN-ARG-ARG-T
RP-ARG-ALA-LYS-VAL-GLN-AS
1 containing N-ARG-ILE- and expressing a peptide similar to the immune response activity or osteoinductive activity of BMP-p
15. The bone morphogenetic peptide agent according to claim 14, wherein the expressed DNA is characterized by a codon sequence that includes one or more base substitutions, additions, deletions, or insertions. (16) The probe has one or more base substitutions,
The amino acid sequence code or amino acid sequence of BMP-p, including additions, deletions, or insertions, is: -ILE-P
RO-GLN-GLN-ARG-ARG-TRP-AR
G-ALA-LYS-VAL-GLN-ASN-ARG
- selecting a DNA sequence that hybridizes to a nucleic acid probe containing a DNA or RNA sequence or its complement that is an ILE, whatever its origin;
A step for selecting a DNA sequence encoding the expression of a peptide exhibiting BMP or BMP-p immunoreactive activity or osteoinductive activity from the group of A sequences. (11) Includes a codon sequence or its complement for part or all of the amino acid sequence of BMP-p, or
Exhibits immunoreactive activity or osteoinductive activity of bone morphogenetic protein (BMP) containing part or all of the amino acid sequences of the probes listed in Table 4, including substitutions, additions, deletions, or insertions of contiguous or multiple bases. A nucleic acid probe that specifically selects for nucleic acid sequences encoding expression of a peptide. (18) A DNA sequence that expresses a bone morphogenic peptide agent and hybridizes to the probe according to claim 17. (19) Includes direct chemical synthesis of DNA containing the codon sequence for the amino acid sequence of BMP-p, including addition, substitution, deletion, or insertion of one or more bases;
or enzymatic synthesis of cDNA from an RNA template containing codons for the amino acid sequence of BMP-p;
A step of producing a DNA sequence encoding the expression of a peptide that exhibits the immunological activity or osteoinductive activity of BMP or BMP-p, which includes the addition, substitution, deletion, or insertion of one or more bases. (20) An expressible set of bone morphogenic agents comprising a method of inducing a DNA sequence selected according to claim 16 or produced according to claim 15 into a cloning vehicle. Method for producing recombinant DNA molecules. (21) DNA selected according to the process of claim 16 or produced according to the process of claim 19
A recombinant DNA molecule that includes a cloning vehicle into which sequences have been inserted. (22) Recombinant DNA according to claim 21
Host transformed with the molecule. (23) Immune reaction of BMP or BMP-p comprising means for transforming a suitable host with a recombinant molecule, means for culturing said host and means for collecting said peptide according to claim 21. A method for producing a bone morphogenetic peptide that exhibits active or osteoinductive activity. (24) Producing a bone morphogenetic peptide that exhibits the immune activity or osteoinductive activity of BMP, including proteolyzing BMP during the production of BMP-p having a relative molecular weight in the range of about 4K to about 7K. how to. (25) The method according to claim 24, wherein the proteolysis of BMP is performed using pepsin. (26) The method according to claim 24, wherein the proteolysis of BMP is performed using trypsin. (27) Immune reaction of BMP, which includes the addition, substitution, deletion, or insertion of one or more amino acids, and includes direct chemical synthesis of the peptide from the peptide containing the constituent amino acids and the amino acid sequence of BMP-p. A method for producing a bone morphogenetic peptide that exhibits active or osteoinductive activity. (28) Includes a bone morphogenetic peptide agent selected from the group consisting of peptides as set forth in any one of claims 1 to 8 and 13 to 15. Ingredients for inducing bone formation. (29) A method of inducing bone formation in a vertebrate, comprising administering to the vertebrate an effective amount of the component according to claim 28 in a pharmaceutically acceptable manner.